Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in Hela cells

Abstract

Patients with the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) or familial partial lipodystrophy (FPLD) have specific mutations in the lamin A gene. Three such point mutations, G465D (FPLD), R482L, (FPLD), or R527P (EDMD), were introduced by site-specific mutagenesis in the C-terminal tail domain of a FLAG-tagged full-length lamin A construct. HeLa cells were transfected with mutant and wild-type constructs. Lamin A accumulated in nuclear aggregates and the number of cells with aggregates increased with time after transfection. At 72 h post transfection 60–80% of cells transfected with the mutant lamin A constructs had aggregates, while only 35% of the cells transfected with wild-type lamin A revealed aggregates. Mutant transfected cells expressed 10–24×, and wild-type transfected cells 20×, the normal levels of lamin A. Lamins C, B1 and B2, Nup153, LAP2, and emerin were recruited into aggregates, resulting in a decrease of these proteins at the nuclear rim. Aggregates were also characterized by electron microscopy and found to be preferentially associated with the inner nuclear membrane. Aggregates from mutant constructs were larger than those formed by the wild-type constructs, both in immunofluorescence and electron microscopy. The combined results suggest that aggregate formation is in part due to overexpression, but that there are also mutant-specific effects.

Introduction

Mutations in the lamin A gene have been shown to be responsible for six human diseases. The myopathies include dilated cardiomyopathy (DCM) with conduction system defects [1], limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances (LGMD1B) [2], and the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) [3], [4]. This latter disease is characterized by early contractures of the ankles, elbows, and Achilles’ tendons, slowly progressive muscle wasting, and weakness and in some cases cardiomyopathy with conduction block [5]. The fourth disease is Dunnigan-type familial partial lipodystrophy (FPLD) [6], [7], [8]. Patients suffering from FPLD show complete or partial absence of adipose tissues from the extremeties and trunk after puberty [9]. Additional diseases associated with mutations in the lamin A gene are autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2) [10] characterized by muscle weakness and wasting, foot deformities, and electrophysiological as well as histological changes, and mandibuloacral dysplasia, a rare autosomal recessive disorder characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation [11]. Mutations causing each of the above diseases have been mapped along the lamin A gene (reviewed by Genschel and Schmidt [12] and Hutchison et al. [13]).

Lamins A and C arise from alternatively spliced forms of the lamin A gene and diverge only at their C-termini [14], [15], [16]. Expression of the lamin A/C gene is developmentally regulated. In mice lamin A/C expression is not found in cells of the early embryo or in undifferentiated teratocarcinoma cell lines. It is detected in the embryo proper first at embryonic day 12 in certain muscle cells, but begins to be expressed only at 15 days after birth in brain [17], [18]. Stem cells of the immune and hemopoietic systems lack lamin A/C even in adult animals [19]. In contrast, lamins B1 and B2 are encoded by two distinct genes and are thought to be constituitively expressed irrespective of developmental stage [20], [21], [22] and, indeed, B-type lamins are found at all stages of mouse development [18]. A third B-type lamin—lamin B3—is a splice variant of lamin B2 and its expression is limited to male germ cells [23].

Knockout of the lamin A gene in mice results in mice that at birth cannot be distinguished from wild-type, except for some redistribution of emerin, a protein of the inner nuclear membrane. However, such mice show severely retarded postnatal growth, develop muscular dystrophy, and die at 6–8 weeks of age. Thus, the pathology of the lamin A knockout mice is strikingly similar to that seen with human EDMD [24]. RNA interference experiments on lamins in human HeLa cells have shown that silencing of the lamin A gene did not affect cell growth, but did lead to a redistribution of emerin. These results show that lamin A is not an essential protein in HeLa cells. In contrast, lamin B1 and lamin B2 are essential since silencing of these genes leads to apoptosis [25].

The nuclear lamina underlies the inner membrane of the nuclear envelope [26]. This 10–20-nm layer is built of polymers formed by the nuclear lamins, which are most probably arranged as a net, although this ultrastructural feature has so far only been demonstrated for the Xenopus oocyte [27]. It is thought to play a critical role in maintaining the structural integrity of the nuclear envelope, but may also have additional functions. The lamina is connected to the nuclear envelope by lamin-binding proteins that are integral membrane proteins of the inner nuclear membrane. This latter class includes three isoforms of lamina-associated protein 1 (LAPI A, B, and C), at least five isoforms of LAP2, emerin, and the lamin B receptor [28], [29]. A sixth isoform of LAP2, LAP2α, lacks an inner nuclear membrane-binding domain [28]. Emerin binds specifically to A-type lamins both in vivo [30], [24] and in vitro [31]. LAP1A and LAP1B specifically bind to lamin A, C, and B1 [32], while LAP2α is associated exclusively with A-type lamins [33] and lamin B receptor interacts with B-type lamins [34]. The lamina is also associated with nuclear pore complexes perhaps through a tight association of nuclear lamins with Nup153, a peripheral nuclear pore component [35]. Interactions between the nuclear lamina and chromatin are thought to be mediated through the C-terminal lamin sequences [36].

To understand how different mutations in the lamin A molecule can affect lamina assembly and nuclear organization, we and others have used additional approaches with cells in culture. In the first approach, primary cultures of fibroblasts from FPLD patients with heterozygous mutations in lamin A at position 482 were studied [37]. Dysmorphic nuclei were seen in fibroblasts from three patients. The frequency with which such abnormalities were observed varied between 5% and 22%, depending on the patient and the passage number, while 2–4% of control fibroblasts had similar abnormalities. Equivalent studies have not yet been performed with cells from patients with the other three diseases. In a second approach, point mutations were inserted into the full-length lamin A cDNA and used to transfect cells in culture [38], [39]. Changes were seen in the lamin A arrangement after transfecting HeLa cells with two point mutants that cause DCM and a single mutant that causes EDMD, but not with a point mutation causing FPLD [38]. In a second study, aggregate formation was seen in the lamin A arrangement after transfecting C2C12 myoblasts with four mutants, one of which causes DCM and three of which cause EDMD [39]. No change was seen in the same study with 11 other mutant constructs, three of which cause DCM, five of which cause EDMD, and three cause FPLD. Our own interest in lamin A led us to make several mutant lamin A constructs that cause EDMD and FPLD in parallel to the studies reported by Östlund et al. [39] and Raharjo et al. [38]. Our results suggest that, as argued by others, there are mutant-specific effects. In addition, however, they give insight into lamin A aggregate formation. This phenomenon was reported by Östlund et al. [39] with mutant, but not with the wild-type, lamin A construct. Our data show that both the mutant and the wild-type lamin A constructs form aggregates. At any time point the mutant cultures have about twice as many transfected cells with aggregates as do the wild-type cultures. Other lamins, as well as several lamin-associated proteins, are recruited into the aggregates. Aggregates have been characterized not only by immunofluorescence microscopy but also by electron microscopy, and the majority are shown to underlie the nuclear membrane.